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Learn about Immunoprecipitation which is a technique that uses antibodies specific to a protein to remove those proteins from solution. The antibody-protein complexes are precipitated out of solution with the addition of an insoluble form of antibody binding proteins.

Immunoprecipitation

Immunoprecipitation is a technique that uses antibodies specific to a protein to remove those proteins from solution. The antibody-protein complexes are precipitated out of solution with the addition of an insoluble form of antibody binding proteins. Examples include Protein A, Protein G, Zysorbin, Immunoprecipitin, or the addition of a second antibody to the solution (see diagram 1 below).

Samples can then be washed after precipitation and centrifugation. The precipitates can then be run on SDS-PAGE gels with protein sample buffer. Usually the protein samples are radiolabeled prior to cell lysis. This is done by usually adding radiolabeled amino acids to cells. This makes things simple as the protein is then easily detected on film when run on a gel as the spot will emit radioactivity and expose film. You can then assess for the size of the protein (in molecular weight with comparison to size standards), and quantity (by comparing treated samples or to a standard of quantities).

Moreover, the immunoprecipitation procedure also allows the concentration of proteins that are not very abundant or difficult to detect using western blot. IP can concentrate proteins up to 10,000-fold as it can take large volumes of cell lysates and concentrate your protein of interest into a small precipitate which can then be used for western blot analysis or other methods.

Applications of Immunoprecipitation:

1) Determination of the molecular weight and quantity of immunoprecipitated protein by SDS-PAGE.

2) Immunoprecipitation is used to assess for protein-protein interactions. This is done by immunoprecipitation for one protein, and then blotting for another protein.

3) Quantification of rate of synthesis of a protein in cells by determining the quantity radiolabeled protein made during a specific amount of time.

4) Immunoprecipitation enables to concentration of proteins that are otherwise difficult to detect.

The immunoprecipitation method can be divided into several steps:

Washing of the antibody-antigen complexes to remove non-specific proteins and contaminants.

Analysis of complexes/antigen of interest by SDS-PAGE

Immunoprecipitation Sample Preparation and Cell Lysis

Immunoprecipitation Lysis Buffers

Depending on the application of immunoprecipitation or IP, you will want to use different lysis buffers. The choice of lysis buffer is important and is dependent on the characteristics of the protein of interest.

If want to study phosphorylation of proteins or protein-protein interactions, you should try Np-40.

If you are studying total protein levels of a protein, you should try RIPA.

RIPA buffer gives lower background in immunoprecipitation. However, RIPA can denature some proteins. If you are conducting immunoprecipitation experiments to study protein-protein interactions, RIPA should not be used as it can disrupt protein:protein interactions.

NP-40 buffer has a lower deanturing level, and is thus used for phosphorylation experiments such as looking at kinase activity. Also NP-40 is used for protein-protein interactions. NP40 is a non-ionic detergent, and is the most commonly used detergent in cell lysis buffers for immunoprecipitation and western blot.

RIPA Cell Lysis Buffer - Immunoprecipitation Buffer Recipe

50 mM Tris-HCl

Adjust to pH 7.4

150 mM NaCl

1 mM PMSF

1 mM EDTA

5 µg/ml Aprotinin

5 µg/ml Leupeptin

1% Triton x-100

1% Sodium deoxycholate

0.1% SDS

NP-40 Cell Lysis Buffer - Immunoprecipitation Buffer Recipe

50 mM Tris-HCl Final Concentration

150 mM NaCl Final Concentration

Add 1% NP-40

Adjust to pH 8.0

Pre-clearing for Immunoprecipitation

The preclear step is usually done for immunoprecipitation because it lowers the amount of non-specific proteins in the cell lysate. Furthermore, pre-clearing also removes proteins that may have possible non-specific interactions and a high affinity for Protein A, Protein G, Immunoprecipitin, Zysorbin, or whatever insoluble form of antibody binding protein you are using to pull-down your antibody. Some researchers find this step un-necessary and skip this step. Try pre-clearing the first time you try immunoprecipitation. If your results are quite clear, try skipping pre-clearing next experiment and see how it turns out.

IP: Incubation Antibody and Formation of Antibody-Antigen Complexes

Which antibody should you use for immunoprecipitation? Usually Polyclonal antibodies are used for immunoprecipation.

Immunoprecipitation - Precipitation of the Complex of Interest

The antibody-antigen complexes are not insoluble by themselves, ie. immunoprecipitation, is not a complete idea. Antibodies and their binding to antigens are soluble in blood, buffers, and during the immunoprecipitation experiment. What is used to precipitate out these complexes is insoluble antibody binding proteins. Proteins A and G, were isolated from bacteria, and bind to the constant region of the antibody.

Examples of insoluble antibody binding proteins used to precipitate complexes in immunoprecipitation are:

Protein A

Protein G

Zysorbin

Immunoprecipitin

Immunoprecipitation - Washing Steps

Washing the complexes is done using RIPA, PBS, IP-wash buffer, or other buffers depending on what you would like to analyze after IP. RIPA buffer is more stringent whereas PBS is less stringent.

IP Wash Buffer Recipe

10mM Tris; Adjust to pH 7.4

1mM EDTA

1mM EGTA; pH 8.0

150mM NaCl

1% Triton X-100

0.2mM sodium ortho-vanadate

protease inhibitor cocktail

Final Notes for Immunoprecipitation

Immunoprecipitation success is dependent on the affinity of the antibody for its antigen. A good immunoprecipitation antibody will give you low background when you analyze the samples using other techniques after IP. The insoluble antibody-binding proteins used are also very important. Polyclonal antibodies are the best antibodies to use however purified monoclonal antibodies can also be used. Check your antibody vendor to see if your monoclonal antibody was tested for immunoprecipitation.

Tips for Immunoprecipitation

Use 2 mL tubes for immunoprecipitation as they precipitates are more easily washed and resuspended

Try skipping the pre-clearing step to save time

Instead of washing with IP-wash buffer try PBS

If you already have low background and your antibody is great, try ip washing 2X instead of 5X

Make sure you remove as much liquid from the eppendorf tubes when immunoprecipitation washing